The present invention relates to adeno-associated virus (AAV) helper function systems for use in recombinant AAV (rAAV) virion production and methods of using such systems. More specifically, the present invention relates to AAV helper functions that produce low amounts of the long form of AAV Rep proteins, resulting in high-efficiency production of rAAV.
Scientists are continually discovering genes that are associated with human diseases such as diabetes, hemophilia and cancer. Research efforts have also uncovered genes, such as erythropoietin (which increases red blood cell production), that are not associated with genetic disorders but code for proteins that can be used to treat numerous diseases. However, despite significant progress in the effort to identify and isolate genes, a major obstacle facing the biophannaceutical industry is how to safely and persistently deliver effective quantities of these genes"" products to patients.
Currently, the protein products of these genes are synthesized in cultured bacterial, yeast, insect, mammalian, or other cells and delivered to patients by intravenous injection. Intravenous injection of recombinant proteins has been successful but suffers from several drawbacks. First, patients frequently require multiple injections in a single day in order to maintain the necessary levels of the protein in the blood stream. Even then, the concentration of protein is not maintained at physiological levelsxe2x80x94the level of the protein is usually abnormally high immediately following injection and far below optimal levels prior to injection. Second, intravenous delivery often cannot deliver the protein to the target cells, tissues or organs in the body. And, if the protein reaches its target, it is often diluted to non-therapeutic levels. Third, the method is inconvenient and severely restricts the patient""s lifestyle. The adverse impact on lifestyle is especially significant when the patient is a child.
These shortcomings have led to the development of gene therapy methods for delivering sustained levels of specific proteins into patients. These methods allow clinicians to introduce DNA coding for a gene of interest directly into a patient (in vivo gene therapy) or into cells isolated from a patient or a donor (ex vivo gene therapy). The introduced DNA then directs the patient""s own cells or grafted cells to produce the desired protein product. Gene delivery, therefore, obviates the need for daily injections. Gene therapy may also allow clinicians to select specific organs or cellular targets (e.g., muscle, blood cells, brain cells, etc.) for therapy.
DNA may be introduced into a patient""s cells in several ways. There are transfection methods, including chemical methods such as calcium phosphate precipitation and liposome-mediated transfection, and physical methods such as electroporation. In general, transfection methods are not suitable for in vivo gene delivery. There are also methods that use recombinant viruses. Current viral-mediated gene delivery methods include retrovirus, adenovirus, herpes virus, pox virus, and adeno-associated virus (AAV) vectors. Of the more than 100 gene therapy trials conducted, more than 95% used viral-mediated gene delivery. C. P. Hodgson, Bio/Technology 13, 222-225 (1995).
One viral system that has been used for gene delivery is adeno-associated virus (AAV). AAV is a parvovirus which belongs to the genus Dependovirus. AAV has several attractive features not found in other viruses. First, AAV can infect a wide range of host cells, including non-dividing cells. Second, AAV can infect cells from different species. Third, AAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration. Indeed, it is estimated that 80-85% of the human population has been exposed to the virus. Finally, AAV is stable at a wide range of physical and chemical conditions which lends itself to production, storage and transportation requirements.
The AAV genome is a linear, single-stranded DNA molecule containing 4681 nucleotides. The AAV genome generally comprises an internal non-repeating genome flanked on each end by inverted terminal repeats (ITRs). The ITRs are approximately 145 base pairs (bp) in length. The ITRs have multiple functions, including as origins of DNA replication and as packaging signals for the viral genome.
The internal non-repeated portion of the genome includes two large open reading frames, known as the AAV replication (rep) and capsid (cap) genes. The rep and cap genes code for viral proteins that allow the virus to replicate and package the viral genome into a virion. In particular, a family of at least four viral proteins are expressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular weight. The AAV cap region encodes at least three proteins, VP1, VP2, and VP3.
AAV is a helper-dependent virus; that is, it requires co-infection with a helper virus (e.g., adenovirus, herpesvirus or vaccinia) in order to form AAV virions. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome, but infectious virions are not produced. Subsequent infection by a helper virus xe2x80x9crescuesxe2x80x9d the integrated genome, allowing it to replicate and package its genome into infectious AAV virions. While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV will replicate in canine cells co-infected with a canine adenovirus.
AAV has been engineered to deliver genes of interest by deleting the internal non-repeating portion of the AAV genome and inserting a heterologous gene between the ITRs. The heterologous gene is typically functionally linked to a heterologous promoter (constitutive, cell-specific, or inducible) capable of driving gene expression in the patient""s target cells under appropriate conditions. Termination signals, such as polyadenylation sites, can also be included.
To produce infectious rAAV containing the heterologous gene, a suitable producer cell line is transfected with an AAV vector containing a heterologous gene. AAV helper functions and accessory functions, which are typically derived from a helper virus such as adenovirus, are then expressed in the producer cell. Once these factors come together, the heterologous gene is replicated and packaged as though it were a wild-type AAV genome, forming a recombinant virion. When a patient""s cells are infected with the resulting rAAV virions, the heterologous gene enters and is expressed in the patient""s cells. Because the patient""s cells lack the rep and cap genes and the adenovirus accessory function genes, the rAAV are replication defective; that is, they cannot further replicate and package their genomes. Similarly, without a source of rep and cap genes, wild-type AAV cannot be formed in the patient""s cells.
In the earliest attempts to generate rAAV virions, researchers cotransfected an AAV vector carrying heterologous DNA with a wild-type AAV genome. Alternatively, cells transfected with an AAV vector were infected with wild-type AAV particles. In both types of protocols, accessory functions were provided by infection with a helper virus such as adenovirus. The wild-type AAV genome provides the necessary rep and cap functions, but significant amounts of wild-type AAV are produced by these methods. Moreover, the rAAV titers produced are usually not sufficient for therapy.
In order to increase viral titers, several groups have focused their attention on Rep protein expression. For example, one group of researchers constructed a plasmid containing the rep and cap genes linked to an SV40 origin of replication. See, e.g., U.S. Pat. No. 5,693,531. The SV40 origin of replication allows high copy number episomal replication of the plasmid in cells that express SV40 large T antigen. As a result, large amounts of Rep and Cap gene products are produced.
Many other researchers have replaced the AAV p5 promoter with heterologous promoters and enhancers in an effort to increase rAAV titers. For example, one group of researchers replaced the p5 promoter with the HIV LTR. U.S. Pat. No. 5,658,776. In 293 cells, which express the adenoviral E1A gene product, the HIV LTR provides strong constitutive expression of the Rep gene products. The authors emphasize that use of the HIV LTR avoids the problem of Rep autoregulating expression from the p5 promoter, and conclude that in previously used methods, levels of Rep expression are limiting in the production of rAAV.
All of these approaches reflect a widely held notion that expressing large amounts of the Rep gene products will allow the production of high titer rAAV. The long forms of Rep, however, have toxic effects on many cell types.
From the foregoing, it will be appreciated that it would be an advancement in the art to provide AAV helper functions that allow high efficiency production of rAAV. It would be a further advancement in the art to provide such helper functions that produce only small quantities of the long forms of the Rep proteins.
Such AAV helper functions and methods of their use are disclosed herein.
The present invention relates to AAV helper function systems for rAAV production and methods of using such systems. In certain embodiments, a method of the present invention includes the steps of (1) introducing an AAV vector into a suitable host cell; (2) introducing an AAV helper function vector into the host cell such that the host cell produces only small amounts of the long forms of Rep protein; (3) expressing accessory functions in the host cell; and (4) culturing the host cell to produce rAAV virions. In certain preferred embodiments, the amount of the long forms of Rep protein produced are substantially less than the amount that would be produced in the host cell using an AAV helper function vector expressing the long forms of Rep protein under the control of an AAV promoter. In certain other embodiments, the amount of the long forms of Rep protein produced are less than the quantities of the short forms of Rep protein produced by the host cell. It will be appreciated that both transcriptional and translational controls may be used to limit the levels of the long forms of Rep protein that are produced.
The AAV vector and AAV helper function vector can be transfected into the host cell, either sequentially or simultaneously, using well-known techniques. Accessory functions may be expressed in any of several ways, including infecting the host cell with a suitable helper virus (such as adenovirus, herpesvirus, or vaccinia virus), or by transfecting one or more accessory function vectors into the host cell. It is also well known in the art that certain cell lines, e.g., 293 cells, inherently express one or more accessory functions.
An AAV helper function vector of the present invention may be a plasmid, bacteriophage, transposon, cosmid, chromosome, artificial chromosome, virus, or other suitable genetic element, and may include selectable genetic markers such as antibiotic resistance genes. Such vectors may also include one or more accessory function genes, such as the E1A, E1B, E2A, VA RNA, and E4 regions of adenovirus.
Also provided herein are host cells for producing rAAV virions. In certain embodiments, a host cell of the present invention comprises an AAV helper function vector that directs the host cell to produce only small amounts of the long forms of Rep protein. Upon introduction of an AAV vector and expression of accessory functions in the host cell, rAAV virions are produced. In certain preferred embodiments, a host cell of the present invention also includes one or more accessory functions.
The rAAV virions produced using the present invention may be used to introduce genetic material into animals, including humans, or isolated animal cells for a variety of research and therapeutic uses. For example, rAAV virions produced using the methods of the present invention may be used to express a protein in animals to gather preclinical data or to screen for potential drug candidates. Alternatively, the rAAV virions may be used to transfer genetic material into a human to cure a genetic defect or to effect a desired treatment.
These and other objects and advantages of the present invention will become apparent upon reference to the accompanying drawing and upon reading the following detailed description and appended claims.